Costimulatory signals play crucial roles in T cell biology as they determine the outcome of T cell receptor signaling (see Chen et al., Nat Rev Immunol 13: 227-242 (2013)). Blocking of the costimulatory protein-protein interaction is one of the most actively investigated pathways to mitigate immune responses in transplant patients and autoimmune diseases. According to the current knowledge, T cell activation is thought to require at least two signals: (1) engagement of the T cell receptor (TCR) with the MHC-peptide complex and (2) ligation of costimulatory molecules on T cells with their respective ligands on antigen-presenting cells (APCs). T cells receiving signal 1 and positive costimulation undergo proliferation, cytokine production, and further differentiate into effector cells. Even if the underlying mechanisms are not entirely understood, it is generally believed that antigen recognition in the absence of costimulation may alter the immune response and ultimately lead to tolerance.
Several cell surface receptor-ligand pairs provide important co-signaling (costimulatory as well as coinhibitory signaling) interactions that regulate T cell activation. The proteins involved in these cell surface interactions belong to two main families: the immunoglobulin superfamily (CD28-CD80/86 and ICOS-ICOS-L) and the tumor necrosis factor (TNF)-TNFR superfamily. Costimulation of T-cell activation has been reported for several members of the TNFR-TNF superfamily (see Locksley et al., Cell 104:487-501 (2001); Croft, Nat Rev Immunol 3:609-620 (2003); Watts, Annul Rev Immunol 23:23-68 (2005); Li et al., Immunol Rev 229:271-293 (2009); and Croft, Nat Rev Immunol 9:271-285 (2009)), and all its members play important roles in various aspects of the immune response (see Locksley et al., Cell 104:487-501 (2001); Aggarwal, Nat Rev Immunol 3:745-756 (2003); and Yao et al., Nat Rev Drug Discov 12:130-146 (2013)). Consequently, they are promising therapeutic targets in autoimmune diseases, in transplant recipients as well as in cancers (see Chen et al., Nat Rev Immunol 13: 227-242 (2013); Locksley et al., Cell 104:487-501 (2001); Aggarwal, Nat Rev Immunol 3:745-756 (2003); and Yao et al., Nat Rev Drug Discov 12:130-146 (2013)). Costimulatory blockade has emerged as a particularly valuable target for immune modulation both in transplant recipients and in autoimmune diseases since it might avoid the broad suppression of immunity caused by currently existing immunosuppressive agents (see Li et al., Immunol Rev 229:271-293 (2009); Gao et al., Diabetes Metab Res Rev 19:179-185 (2003); Larsen et al., am J Transplant 6:876-833 (2006); Vinceti et al., Annu Rev Med 58:347-358 (2007); Weaver et al., Front Biosci 13:2120-2139 (2008); and Peters et al., Semin Immunol 21:293-300 (2009)). Because members of this family are expressed only upon T-cell activation (with the exception of CD27), they are generally considered to play a particularly important role in the effector and memory phases of the immune response (see Li et al., Immunol Rev 229:271-293 (2009)).
The TNF superfamily (TNFSF) contains about thirty structurally related receptors (TNFSF-R) and about twenty protein ligands that bind to one or more of these receptors (see Locksley et al., Cell 104:487-501 (2001); Aggarwal, Nat Rev Immunol 3:745-756 (2003); Bodmer et al., Trends Biochem Sci 27:19-26 (2002); Bossen et al., J Biol Chem 281:13964-13971 (2006); Tansey et al., Drug Discov Today 14:1082-1088 (2009); and Croft et al., Nat Rev Drug Discov 12:147-168 (2013)). TNFSF ligands are soluble or membrane-anchored trimers that cluster their cell surface receptors to initiate signal transduction. These interactions are integral to communication and signaling systems involved in numerous physiological functions essential to inflammatory signaling, to the functioning of the immune and nervous system, to bone development, and others. There are biologics in clinical development for almost all of these interaction pairs (see Tansey et al., Drug Discov Today 14:1082-1088 (2009); and Croft et al., Nat Rev Drug Discov 12:147-168 (2013)). Currently, there are five biologics blocking TNF (TNFSF2) or LTα (TNFSF1) that are approved for treating various autoimmune and inflammatory disorders including rheumatoid arthritis (RA), psoriatic arthritis, juvenile idiopathic arthritis, psoriasis, ankylosing spondylitis, Crohn's disease and ulcerative colitis: etanercept (LTα, TNFSF1 and TNF, TNFSF2), infliximab, adalimumab, certolizumab pegol, and golimumab (TNF, TNFSF2). There are also biologics targeting other TNFSF members approved for clinical use: brentuximab vedotin (CD30L, TNFSF8) for Hodgkin's lymphoma and systemic anaplastic large cell lymphoma (sALCL); denosumab (RANKL, TNFSF11) for osteoporosis, and belimumab (BAFF, TNFSF13B) for systemic lupus erythematosus (SLE) and RA (see Croft et al., Nat Rev Drug Discov 12:147-168 (2013)). Within the TNF superfamily, the CD40-CD40L interaction is one of the most important and most extensively studied interactions. CD40 (TNFRSF5) and CD40L (CD154, TNFSF5) were, in fact, the first TNFSF costimulatory molecules to be identified. CD40-CD40L is a therapeutic target in many diseases due to its involvement in driving inflammatory events and autoimmunity, and the therapeutic effects of its inhibition are mainly due to the suppression of T and B cell mediated immune responses. Blocking of this protein-protein interaction is a proven highly effective immunomodulatory therapy (see Burkley, Adv Exp Med Biol 489:135-152 (2001); Quezada et al., Annu Rev Immunol 22:307-328 (2004); Daoussis et al., Clin Diagn Lab Immunol 11:635-641 (2004); and Elgueta et al., Immunol Rev 229:152-172 (2009)). Hence, there is considerable ongoing interest in targeting CD40L and/or CD40, and multiple antibodies have been developed and reached different phases of preclinical or clinical testing. Corresponding biologics in clinical development include PG102, ASKP1240/4D11, lucatumumab (HCD122), dacetuzumab (SGN 40), Chi Lob 7/4, and CP 870893 (see Croft et al., Nat Rev Drug Discov 12:147-168 (2013)).
Such antibodies (immunoglobulins) have the advantage of being highly specific for their targets and quite stable in human serum; however, they usually cannot reach intracellular targets (see Verdine et al., Clin Cancer Res 13:7264-7270 (2007)) and, as all other protein therapies, are often hindered by solubility, route of administration, distribution, and stability problems as well as by the possibility of a strong immune response mounted against them (see Leader et al., Nat Rev Drug Discov 7:21-39 (2008)). For immunomodulatory biologics, a high incidence of additional unwanted adverse reactions (including serious infections, malignancy, cytokine release syndrome, anaphylaxis, hypersensitivity as well as immunogenicity) always looms as a hindrance in their development (see Sathish et al., Nat Rev Drug Discov 12:306-324 (2013)). While some of these are due to on-target interactions (i.e., exaggerated pharmacology), some (e.g., generation of neutralizing anti-drug antibodies and hypersensitivity reactions) are due to the inherent immunogenicity of biologics (see Sathish et al., Nat Rev Drug Discov 12:306-324 (2013)). For example, out of the 40 licensed immunomodulatory biologics, 18 have been associated with serious infections, including reactivation of bacterial, viral, fungal, and opportunistic infections. Traditional small-molecule drugs could provide a convenient alternative. However, small molecules were not pursued at all as possible PPI inhibitors for a long time as they were considered unlikely to be effective. This is because these interactions usually involve relatively large protein surfaces (1,500-3,000 Å2) that also lack the well-defined binding pockets present on traditional targets of most existing drugs (G-protein coupled receptors—GPCRs, ion channels, enzymes) (see Buchwald, IUBMB Life 62:724-731 (2010)). During the last two decades, it has become increasingly clear that, at least in certain cases, small molecules can act as effective PPI modulators. Sufficiently effective small-molecule inhibitors have been identified for a few important PPIs and several candidates are in advanced clinical development (see Buchwald, IUBMB Life 62:724-731 (2010); Berg, Angew Chem Int Ed Engl 42:2462-2481 (2003); Arkin et al., Nat Rev Drug Discov 3:301-317 (2004); Wells et al., 450:1001-1009 (2007); Fry, Biopolymers 84:535-552 (2006); Whitty et al., Nat Chem Biol 2:112-118 (2006); Mullard, Nat Rev Drug Discov, 11:173-175 (2012); and Arkin et al., 21:1102-1114 (2014)). Intriguingly, some of the latest data suggest that if the initial hurdles can be overcome, small-molecule PPIIs actually tend to perform quite well in clinical development: While very few such PPIIs have made it to clinical trials, those that do have a better than average chance of success (see Meier et al., 18:607-609 (2013)). For example, in phase I, latest-generation PPIIs (those that have been in development between 2005 and 2012) had an 82% probability of success, compared to 54% for all new molecular entities (NMEs), and in phase II, their probability of success was 57% vs. 34% for all NMEs (see Meier et al., 18:607-609 (2013)).
Targeting of TNFSF costimulatory interaction in general and of the CD40-CD40L in particular with small molecule inhibitors is of special interest for those suffering from some autoimmune disease as well as for transplant recipients in general and for pancreatic islet transplant recipients in particular. The modulation of these interactions can be beneficial in pathogenic processes of chronic inflammatory diseases, such as autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis. Autoimmune diseases include diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type I (juvenile) diabetes, mixed connective tissue disease MCTD, Celiac disease, Crohn's disease, Grave's disease, Sjögren's syndrome, dermatomyositis, psoriasis, neurodegenerative disorders, and others. Certain organic dyes and related compounds can effectively interfere with costimulatory protein-protein interactions such as the CD40-CD40L or the OX40-OX40L, and we have identified the first small-molecule compounds capable of modulating these interactions (see Margolles-Clark et al, J Mol Med 87:1133-1143 (2009); Margolles-Clark et al., Chem Biol Drug Des 76:305-313 (2010); Song et al., Br J Pharmacol 171:4955-4969 (2014); and U.S. Patent Application Publication No. US 2011/0065675). However, because of their dye nature, these compounds (e.g., direct red 80, crocein scarlet 7B, mordant brown, and chlorazol violet N) cannot be used as such for therapeutic applications.
Accordingly, there is a need for new small molecule compounds that can modulate TNFSF costimulatory interactions.